The objectives of this ongoing project include the elucidation of (a) the mechanism of uptake of vitamin B6 by different organisms, (b) the interaction of its coenzyme forms, pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), with various apoenzymes to form the corresponding holoenzymes and the effect of structural modification of both coenzyme and apoenzyme on this interaction, and (c) the catalytic mechanism of action of PLP-enzymes. To this end we have isolated six different bacterial PLP-enzymes in homogeneous form and are conducting extensive comparisons of their spectral and catalytic properties in the presence and absence of substrates and substrate analogues, of the conditions necessary for their resolution from and reconstitution with PLP (e.g., how these processes are affected by pH, ionic strength, carbonyl reagents, protein modification, other proteins, etc.). We are also investigating the effects of modification of protein structure on catalytic properties of these enzymes: such modification can be effected either by chemical modification in structure of the coenzyme, or by mutational alteration of the structure of the apoenzyme. In the latter case, the chemical basis for observed differences will be studied by defining whether the effect is on ability to retain the coenzyme, or upon catalytic activity per se; in either case attempts to define the effects of the mutational alteration in terms of amino acid substitutions will be made. Finally we will use isotopically labeled pyridoxine, pyridoxal, and pyridoxamine to determine whether absorption of vitamin B6 occurs via active transport (if so, attempts to isolate a binding protein will be made), and whether differences in activity of these compounds in promoting growth of auxotrophic organisms result from differences in their uptake, or from differences in intracellular metabolism following uptake.